Human and rat variants of glycoprotein CD44 containing an additional extracellular domain associated with metastatic potential

ABSTRACT

A monoclonal antibody (MAb1.1ASML) directed against a surface glycoprotein of the metastasizing rat pancreatic carcinoma cell line BSp73ASML, was used to identify and isolate a complementary DNA (cDNA) clone capable of encoding for a glycoprotein with partial homology to CD44, a presumed adhesion molecule. This clone, which was subsequently designated pMeta-1, contains an additional extracellular domain of 162 amino acids inserted into the CD44 protein between amino acid positions 223 and 247 (by analogy to human and murine CD44). This new variant was expressed only in the metastasizing cell lines of two rat tumors, the pancreatic carcinoma BSp73 and the mammary adenocarcinoma 13762NF; it was not expressed in non-metastasizing tumor cell lines nor in normal rat tissues. Overexpression of pMeta-1 in the nonmetastasizing BSp73AS cell line suffices to establish full metastatic behavior. The variant-specific rat CD44 sequence was used to isolate a cDNA clone encoding for a human homologue as well.

This application is a division of application Ser. No. 07/946,497, filed Nov. 9, 1992 now U.S. Pat. No. 5,506,119, which is the national stage of PCT/EP91/00614 filed Mar. 30, 1991.

The invention concerns variant CD44 surface proteins, antibodies against the variant determinants of these proteins, as well as processes for their production, furthermore the DNA sequences which code for these variant protein fragments, as well as the use of these proteins or parts thereof and the antibodies directed against them for the diagnosis and therapy of tumour metastases.

BACKGROUND OF THE INVENTION

The ability to metastase forms the actual life-endangering property of malignant tumour cells. The original primary tumour cells probably acquire this property by a whole series of changes in the course of the tumour progression. As a result of this process, cancer cell variants are continuously detached from the primary tumour mass, penetrate the extracellular matrix and migrate into the lymphatic system or the blood circulation. Often adhering to one another, the metastasing tumour cells are transported in the blood or lymph system, leave the vascular system at other places in order there to penetrate into secondary tissue and form daughter tumours (survey of Hart et al., 1989; Nicolson, 1987). The formation of metastases requires a whole series of interactions of the tumour cells with intercellular matrix and other cells. Almost all of these interactions require cell surface components, such as e.g. the receptors for matrix and lamina, surface-bound proteolytic enzymes, as well as cell adhesion molecules with inclusion of those which cause organ-specific adhesion and thus organ preference of the metastasis, furthermore growth factors and growth factor receptors.

It is known that the membrane proteins differentiate non-metastasing and metastasing tumour cells of the BSp73 rat tumours, demonstrated by antibody reaction (Matzku et al., 1983 and 1989).

SUMMARY OF THE INVENTION

It has now been found that the metastasing BSp73ASML tumour cells contain a surface protein which, in part, corresponds to a known glycoprotein participating in the lymphocyte adhesion and cell-cell and cell-matrix exchange action (designation of the normal glycoprotein in humans: CD44, hermes-1, in the mouse: Ppg-1 and in the rat: HEBF1n). However, the new variant CD44 surface protein differs from these known sequences by an extracellular region (ECR) of 154 amino acids which is introduced between the 220th and 237th amino acid of the human CD44 sequence (or 224th and 239th amino acid of the mouse sequence). This new glycoprotein appears to possess an important role for the cell/matrix or cell/cell binding in the case of the metastasis. Therefore, the production and characterisation of this protein region (ECR) forms one of the tasks of the present invention. The present invention includes isolated Meta-1 surface polypeptide of metastasizing tumor cells comprising an amino acid sequence selected from the group consisting of amino acid residues 224 to 378 of SEQ ID NO:2, amino acid residues 53 to 219 of SEQ ID NO:4 and amino acid residues 77 to 186 of SEQ ID NO:4. By immunisation of mice with membrane proteins which have been obtained from BSp73ASML, spleen cells were produced which form antibodies against the ECR of the variant CD44 surface protein. According to the method of Kohler (1981), these are fused by polyethylene glycol with myeloma cells in order to produce permanent cultures. By means of cloning and selection of those cultures which produce antibodies which react with BSp73ASML but not with the non-metastasing parent form and also not with other non-tumorigenic rat cells, there can be obtained specific antibodies against the new protein part ECR. For the further investigation, a monoclonal antibody was chosen which stains the BSp73ASML cells in the immunofluorescence test especially intensively, which has received the designation mAb1.1ASML (mAb: monoclonal antibody).

In the Western blot test, in a protein hydrolysate from BSp73ASML, there can be determined 4 protein bands with molecular weights of 120,000; 150,000; 180,000 and 200,000 with mAb1.1.ASML, whereas extracts from rat fibroblast cells and non-metastasing rat tumour cells give no significant reaction. It has not yet been possible to determine whether these size differences are due to a different original amino acid sequence or to a differently strong subsequent protein modification. In any case, the epitope recognised by the antibody is contained in all 4 protein species but not in the proteins serving for the control from the non-metastasing BSp73As cells or from normal rat cells.

Isolation of cDNA Sequences which Code for the ECR of the Surface Protein

The monoclonal antibody mAb1.1ASML was used in order to discover the ECR-coding cDNA sequences in a bacterial expression bank. The bank was constructed with the help of PolyA+ RNA from BSp73ASML and the pEX vector system (Stanley & Luzio, 1984). The products coded by the cDNA sequences are found as β-galactosidase fusion proteins with the help of the antibodies. A so-isolated cDNA clone positive for the monoclonal antibody 1.1ASML with the name pEX34 carries a cDNA sequence piece of 167 nucleotides. This cDNA piece was now used in order to pattern through a larger cDNA bank in the vector pSP65, again from BSp73ASML RNA. One of these clones isolated therewith, pM66, served thereto to isolate the total length cDNA clone pMeta-1 with the help of so-called "primer" elongation (starter oligonucleotides) and of the polymerase chain reaction (PCR). Evidence of the total length was obtained with the help of the primary elongation (3207 nucleotides). The colinearity with an RNA from the BSp73ASML tumour was documented by RNase and S1 protection analysis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the structure of a cDNA of the rat surface protein p-Meta-1. Bar "A" represents the extracellular region ("ECR") at positions 941-1108. Bar "B" represents the sequence between positions 1403-1572. Bar "C" represents the sequence between the starting position to position 794. Bar "D" represents sequences at the end of the cDNA clone.

FIG. 2 depicts the sequence of the CD44 protein and DNA from p-Meta-1. The signal peptide is represented by " ". The ECR is represented by "--". The transmembrane region is represented by "\\\\\".

FIG. 3 depicts (a) the nucleotide and protein sequence of a human extracellular region and compares the protein sequence to a rat sequence, and (b) compares protein sequences of rat rMeta-1, human hCD44 and mouse mCD44.

FIG. 4 depicts patterns of RNA from cell lines MTPa, MTC, MTF7, MTA, MTLy, MTLn2 and MTLn3.

FIG. 5 depicts a construct containing pMeta-1 and an SV40 promoter, which was introduced into BSp73As cells. Colonies that were G418-resistant and pMeta-1-expressing were obtained. The RNA from these cells was contacted with CD44-specific sample A or D (see FIG. 1).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The isolation of the cDNAs with the help of the expression in bacteria and the recognition by the antibodies proved that the antibody recognised primary amino acid sequence in the ECR of the surface protein.

ECR-coding Messenger RNA is Expressed in BSp73ASML but not in BSp73AS Cells

Three different sequence samples were produced from the cDNA clones in order to demonstrate specific messenger RNAs by hybridisation. A sample A covers the cDNA region which codes for the ECR (positions: 941-1108). The sample B represents sequences between the positions 1403-1572 and sample C carries sequences from the start up to position 794 from pMeta-L (FIG. 1). Poly A+ RNA of the BSp73 tumour cell line was separated electrophoretically and analysed with the help of RNA transfer hybridisation. Four of the cell lines, which do not metastase, contain no RNA which is homologous to the sample A, whereas RNAs from the metastasing tumour cells strongly react BSp73ASML. In this RNA preparation, the sample A recognises a heterogenous mixture of various RNA sizes between 2.2 and 3.3 kb and a larger RNA species of 4.9 kb. The exclusive expression of the specific membrane proteins recognised by the monoclonal antibody 1.1ASML in the metastasing tumour cell variant is obviously based upon the exclusive expression of the corresponding ECR-coding messenger RNAs. Obviously the complete cDNA clone pMeta-1 with 3.2 kb cannot represent all sequences of this RNA species. It can only represent one species from the heterogenous mixture of RNA sizes. The samples B and C give the same hybridisation pattern as sample A in the separation of the BSp73ASML RNA in any case as far as one can ascertain in the heterogeneity, i.e. these RNA species carry sequences which are complementary to all three samples, A, B and C. In contradistinction to sample A, sample B and C also recognise messenger RNA species in the non-metastasing cell lines. However, the RNA sizes differ clearly from those in BSp73ASML, there are, namely, detected four clearly differentiatable messenger RNA species with the sizes 1.5, 2.0, 2.9 and 4.3 kb. Although these RNA species could be hidden in the heterogenous mixture of the RNAs from BSp73ASML, it is, nevertheless, certain that they do not exist in the same amount in the BSp73ASML. It is decisive that RNA sequences with complementarity to sample A are obviously completely absent in the non-metastasing cells. Therefore, we can carefully conclude that the sequences of the samples A, B and C are contained in the same RNAs in the metastasing tumour, namely, in a manner as though sample A sequences have been spliced into the B-C-positive RNAs and as though this alternative splicing process only occurred in the metastasing cell line.

In order to demonstrate the colinearity between RNA and cDNA and in order to analyse the difference of the RNAs between the BSp73AS and the BSp73ASML cells, S1 nuclease and RNase protection analyses were carried out. The protected DNA or RNA fragments could only be smaller than the total length because they contain 5'-end vector sequences which cannot hybridise with the RNAs from the tumour cells. We consider first the transfer to the 3' side: the transfer of sequences with homology to sample A to those to sample B. Both techniques show a single RNA species in BSp73AS which is colinear with the samples over a wide range. Furthermore, 5' thereof differentiate cDNA or RNA sample, which certainly correspond to RNA sequences from BSp73ASML, from the RNA from the BSp73AS cells. In particular, the RNAs from BSp73ASML contain sequences which protect larger fragments of the samples. The largest fragments correspond to the full length of the DNA or RNA pieces which were offered for the protection analysis. Smaller fragments are also detectable. Since the RNA transfer hybridisations have certainly uncovered a heterogenous mixture of different sizes of RNAs, it is possible that these indicate smaller protected fragments of RNA species which diverge elsewhere from the cDNA, i.e. at positions between the previously detected divergence point and the 5' end of the offered samples. The RNA species are also not detectable in the BSp73AS cells.

We now consider the point of the divergence on the 5' side, thus the transition of sequences which hybridise with the sample C to those which hybridise with the sample A, thus the ECR-coding sequence. The analysis gives corresponding results for the 5' breaking point. RNAs from BSp73AS can protect the offered samples only over a small range. Messenger RNAs from BSp73ASML protect longer fragments. They are, namely, colinear over the whole length of the offered sample. One can thus conclude that the cDNA clone represents pMeta-1 sequences which are distinctive in the metastasing tumour cells BSp73ASML. The 3' and 5' regions are also found in RNAs from BSp73AS. The ECR-coding sequences with the definite transitions, which can be mapped with the help of these above-described techniques, indicate that here an alternative splicing mechanism must be present for the RNA formation. The 5' and 3' breakage points of the transitions to the ECR sequences are marked by arrows in FIG. 1.

The Monoclonal Antibody 1.1ASML Identifies a Variant Form of the Glycoprotein CD44

In order to obtain structural information about the surface protein, all cloned cDNA molecules have been sequenced. The nucleotide sequence of the total length clone pMeta-1 and the amino acid sequences derived therefrom are shown in FIG. 2 (SEQ ID NOS. 1 and 2). The total length cDNA clone traverses 3207 nucleotides (SEQ ID NO:1). The 3' terminus carries a PolyA end, two additional polyadenylation signals lie at positions 2288 and 1743. The first ATG codon follows a consensus initiation sequence and opens a reading frame of 1509 nucleotides, corresponding to 503 amino acids (SEQ ID NO:2). As one should assume for a membrane-standing protein, the first 21 amino acids are hydrophobic and represent a signal peptide. No part of these sequences is hitherto to be found in the data bases. However, we found sequence homology to the recently published data about the lymphocyte homing receptor CD44 (of Pgp-1) (Idzerda et al., 1989; Goldstein et al., 1989; Stamenkovic et al., 1989; Nottenburg et al., 1989; Zhou et al., 1989). The homologies are strictly limited to the 5' and 3' parts of the cDNA with inclusion of non-translated regions and they end at the already above-mentioned points of divergence between the BSp73AS and the BSp73ASML RNA sequences. The total extent between the divergence points (in FIG. 2 characterised by colour markings), thus the whole extent of the metastasis-specific ECR-coding sequence, is not represented in the Pgpl or CD44 sequences. The metastasis-specific glycoprotein obviously represents a variant of the CD44 glycoproteins. It carries, namely, an additional extracellular domain of 156 amino acids and thus an expanded extracellular region of 410 amino acids (less 21 amino acid signal peptide), in comparison with 270 amino acids (also less signal peptide) of the unchanged CD44 glycoprotein. However, in the non-metastasing BSp73AS cells, the unchanged forms of this CD44 family are detected. cDNA sequences of these BSp73AS RNAs have also been cloned and the identity with the metastasis-specific clones outside of the extra domain is demonstrated.

The Expression of the Variant CD44 is Correlated with the Metastatic Potential

In order to test whether the expression of the variant CD44 glycoproteins takes place without exception in the BSp73ASML cells and whether it stands in connection with the metastatic potential of these cells or with the metastatic potential in general, we studied a series of isogenic rat tumour cell lines, namely, the tumour cell lines of the mammary carcinoma system 13762 NF (Neri et al., 1982). We here compare cell lines which have been derived from the parental tumour, namely, the MTPa, MTC, MTF7 and MTA cells (group 1), with cell lines which were established from lymph nodes or lung metastases, namely, MTLy, MTLn2, MTLn3 (group 2). The group 1 cells essentially express the normal CD44 pattern similarly to the RNAs from the BSp73AS cells when one hybridises with sample B. On the other hand, with sample A, there is detected a smaller amount of a diffuse RNA band which has about the size 2.5 kb. On the other hand, the group 2 cells show a completely different RNA pattern. Both samples A and B hybridise with larger RNA species. The sizes resemble those which are detected with BSp73ASML. The similarity is also documented by RNAse and S1 protection analyses. On the basis of these data, we conclude that a change of the splicing pattern of the RNA and the expression of variant CD44 is correlated with the formation of metastases and that the acquired pattern in these metastasing mammary carcinoma cells corresponds very much to those which we have already got to know for the metastasing BSp73ASML cell line. The high molecular proteins-recognised by the antibodies correspond to the two high molecular species of proteins which were detected in the BSp73ASML extracts. In this mammary tumour series, we thus discovered also a metastasis-specific expression of RNA species and of high molecular proteins. That in the group 1, thus the so-called parenteral cell lines, any RNAs were found at all which hybridised with the sample A, thus the ECR-coding sequence, and that we can also see a weak coloration of a protein of 100,000 Dalton with the antibody, we attribute to the fact that the group 1 cells also possess small metastasing ability quite contrary to our original cell line BSp73AS which shows no metastasing behaviour at all.

The Monoclonal Antibody 1.1ASML Inhibits Metastasis Formation in the Rat

In a series of experiments for the metastasis formation of the tumour cell line BSp73ASML in isogenic rats, cells were injected subcutaneously and at different times the monoclonal antibody 1.1ASML was injected intraperitoneally at intervals of two to three days before and after the tumour administration. In the scope of this immunological protocol, it was also determined how the immune response of the rat towards the injected antibody had taken place. There result, namely, anti-mouse immunoglobulin antibodies, as well as also anti-idiotype antibodies. The result of this series of experiments is that the growth and the metastasing of the tumour is considerably delayed by injection of 1.1ASML. This delay permits, in its kinetic, the conclusion that the antibody interferes with a primary process of the metastasing. The experiment shows to us that the protein structure on the surface of the metastasing cells recognised by the antibody has a role in the metastasing process and that therapeutic and diagnostic plans are realistic.

Isolation of the Homologous Human Sequence for the ECR-coding Sequence Part of the Rat cDNA

For human tumour cells in culture, the possibility naturally does not exist as a matter of course of detecting, correspondingly the rat system, whether they also still retain metastasing properties. Experiments with immune-deficient mice make possible only very limited predictions regarding the metastasis potential in the case of humans. Therefore, relatively many tumour cell lines, which have been taken up in culture anywhere in the world at points of time lying a long time ago, would have to be tested for whether they express the sequences which we could detect for the rat metastases. It has been possible to find such a tumour cell line. It originates from a large-cell lung carcinoma of humans and bears the number: LCLC97. In this tumour cell line can be detected three definite RNA species (sizes: 5.5; 3.4 and 2.8 kb) which behave quite corresponding to the RNAs which are detectable in the metastasing tumour cell lines of the rat. They hybridise, namely, not only with the sample A but also with the samples B and C, i.e. that also these human RNA species are identical over wide ranges to the cDNA pMeta-1 (85%).

However, the monoclonal antibody 1.1ASML does not react with this tumour cell, i.e. the piece of protein recognised by the antibody must, in the region of the antigen determinant, differ from the proteins which exist on the surface of the human tumour cells. For the non-reactivity, there suffice already the smallest variations on the basis of the high specificity of the antibody. The human tumour cell LCLC97 now served the purpose of constructing a cDNA bank. On the basis of the high agreement between the rat and human sequences, a cDNA clone could be isolated which showed homology with the sample A. The human cDNA was sequenced. In FIG. 3 (a,b) is shown the primary sequence and the amino acid sequence (SEQ ID NOS 3 and 4) derived therefrom. One can see that, over large regions, identicity exists between the rat (SEQ ID NO:5) and the human sequence. This human sequence (SEQ ID NO:3), as well as also the amino acid sequence (SEQ ID NO:4) derived therefrom, is also the subject of this Patent Specification.

FIG. 3b also shows the protein sequences of surface proteins for rat (rMeta-1, SEQ ID NO:2) human (hCD44, SEQ ID NO:6) and mouse (mCD44, SEQ ID NO:7) with extracellular regions in rat and human protein. rCD44 (SEQ ID NO:8) is also shown in this Figure.

Embodimental Examples Cells and Antibodies

The following cloned Bsp cell lines were used for the investigation: BSp73 14ASML-1 and 10AS-7 and kept in culture as described by Matzku et al., (1983); furthermore, the mammary carcinoma cell lines described by Neri et al., (1982), monoclonal antibodies against BSp73 ASML membrane proteins were produced by immunisation of Balb c mice. After isolation of the spleen cells of an immunised mouse, these were fused with Ag8 Myeloma cells for the immortalisation according to the method for the production of monoclonal antibodies of Kohler (1981). The then-obtained hybridoma cells were subjected to a screening process in order to find those which produce specific antibodies against BSp73ASML but not against BSp73AS and normal rat fibroblast cells. The precise procedure is described in the same way as by Matzku et al. (1989).

Monoclonal antibody (mAb)-producing hybridoma cells with the corresponding specificity were expanded in the tissue culture and the mAb given off into the medium highly enriched by ammonium sulphate precipitation and column chromatography (protein A-Sepharose and MonoQ) and used in this form for the investigations. One of them is mAb1.1ASML.

Immunofluorescence

For the display of the variant CD44 molecule on different tumour cells, these were taken up in culture, then washed with phosphate-buffered common salt solution (PBS) and incubated with 1.1ASML for 30 minutes at 40° C. As secondary antibody for the detection of the binding, there was used a rhodamine-coupled rabbit anti-mouse IgG and shown in the fluorescence microscope.

Construction of the cDNA Expression Banks and Immuno-Screening

PolyA+ RNA from BSp73ASML cells was "primed" with oligo (dT) and hexanucleotides of different composition and synthesised with reverse transcriptase from AMV of the first strand of the cDNA. The second strand of the cDNA was produced with E. coli DNA polymerase I, RNaseH and E. coli ligase and subsequently the double-stranded cDNA linearised on the ends with T4DNA. The vectors pEX1, 2 and 3 (Stanley and Luzio, 1984), which make possible the fusioning of the cDNA in 3 different reading rasters, were cleaved with SmaI restruction endonuclease and ligated with the cDNA (T4 DNA ligase). Competent E. coli DH5 (pCI857) bacteria, which produce a temperature-sensitive repressor, are transfected with the pEX-cDNA constructants and cultured on nylon filters. The gene for the temperature-sensitive repressor RCI857 lies on the plasmid pCI857, which is compatible with the pEX plasmids. At 28° C., the 1P_(R) promotor, which controls the synthesis of the fusion proteins, is inactivated. By temperature increase to 42° C., the CI repressor is inactivated and the synthesis of β-galactosidase/ASML fusion proteins massively set into action. The heat-induced bacteria colonies are subsequently denatured with chloroform vapour on the filters and these then incubated in PBS which contains 3% dry milk powder, lysozyme and DNase. The bacterial fusion proteins fixed on the nylon filter are now incubated with mAb1.1ASML and, after washing out of non-specifically-bound mAb, used for the detection of the binding as secondary antibody 125J-labelled rabbit anti-mouse IgG. After autoradiography, positive clones were isolated from the original bacteria filter and substantially analysed. One clone, which synthesised a fusion protein which reacted specifically with 1.1ASML, was pEX34. The pEX contained in the bacterial clone carries 167 nucleotide cDNA which, inter alia, codes for the epitope (or the antigen determinants), the specificity of which is carried by mAb1.1ASML.

The isolation of the total length cDNA mMeta-1 then took place according to standard methods.

Immunisation of the Rats with mAb1.1ASML

BDX rats, which are syngenic to the BSp73 tumour cells, were injected subcutaneously or intraperitoneally with mAb1.1ASML (coupled to keyhole limpet haemocyananine), together with complete Freund's adjuvant. The first took place 10, 7 and 3 days before the injection of the BSpASML cells (into the fatty foot pad), the following then 3, 7, 11, 14 and 21 days thereafter. After 28 days, the rats were sacrificed, the various lymph nodes prepared and weighed and macroscopically visible lung metastases counted.

Connection between the Expression of Variant CD44 Surface Proteins and Metastatic Potential

In order to ascertain whether the expression of variant CD44 glucoproteins is merely a property of the investigated BSp73ASML cell line or whether the expression can be brought into connection with the metastatic potential, another series of rat tumour cells, which are derived from the 13762NF mammary carcinoma (Neri et al., 1982), were investigated. Furthermore, cell lines which were derived from the primary tumours (MTPa, MTC, MTF7 and MTA (group 1)) were compared with cell lines which are derived from lymph nodes and lung metastases (MTLy, MTLn2, MTLn3 (group 2)). The pattern of the RNA derived from CD44 is given in FIG. 4, whereby samples A, B and D correspond to the samples described on pages 5 and 6 of the Application, as well as to FIG. 1. Cells of group 1 all show a normal CD44 pattern with sample B. However, cells of group 2 show a pattern different therefrom. The RNA is larger than the RNA of group 1 and corresponds to the RNA of BSp73ASML. Smaller RNAs are lost in the case of the hybridisation with sample D. The other patterns show the similarity between the two rat tumour systems.

Also with the sample A, the RNA pattern of group 2 corresponds to that of BSp73ASML. Whereas sample A does not hybridise with RNA from BSp73AS, there is shown a small diffuse RNA band of about 2.5 kb in the case of cells of group 1. RNase and S1 protection analysis also show the structural similarity. From these results, an exchange in the cleavage pattern and the expression of variant CD44 RNAs appears to take place with the formation of metastases.

Transfer of the Metastatic Potential to Non-Metastasing BSp73AS Cells by Overexpression of pMeta-1

The connection of the expression of variant CD44 species with the metastatic potential in two series of rat tumours indicates a causal role of the glycoproteins in the metastatic process. In order to investigate this, pMeta-1 was transferred into BSp73AS cells and investigated whether the behaviour of the cells is thereby changed. The complete coding region of the pMeta-1 (FIG. 2 (SEQ ID NO:1) was inserted below the SV40 promotor and this formation (diagram in FIG. 5) introduced into the BSp73AS cells, together with PSV2neo. Individual G418-resistant and pMeta-1-expressing colonies were obtained. The RNA pattern of 2 of these colonies is shown in FIG. 5. The hybridisation of the variant CD44-specific sample A shows a dominant transcript of approximately 2.2 kb which corresponds to the size of the smallest frequent RNA which is transcribed in BSp73ASML cells (FIG. 5). However, the transfected cells contain about 10 times as much of this RNA as BSp73ASML.

Other size orders are observed in one of the transfected cells (BSp73AS-pSVMeta-1-14), which could be dependent upon the place of the plasmid integration. A pSV2neo simulation transfer clone (not shown) and the BSp73AS receiver cells contain no RNA which is complementary to sample A. In order to discover the endogenic normal CD44 transcriptions (without the extra domains of the pSVMeta-1) in the transfects, the filter was stripped and rehybridised with sample D. This part of the non-transferred 3' sequence is not contained in the expression clone (cf. FIG. 5). Sample D detects two main transcripts of 2.9 and 4.9 kb in the RNA of the two transfects (FIG. 5, right column), not only in the control BSp73AS but also in the non-illustrated BSp73ASpSVneo.

Approximate quantifications of the various agreeing hybridisations show that the transfects express approximately 5× as much of the variant CD44 RNAs, which are transcribed by the expression plasmid, as the endogenic gene transcripts.

The overexpressed cDNA is transferred into a protein. The two transfects, which are illustrated in FIG. 5, synthesise mAb1-immune-colourable proteins of the same apparent size, namely, a main product of 150 kDa and a weaker band at 100 kDa. Since the cDNA sequence codes a primary protein product of only 503 amino acids (corresponds to about 60,000 Dalton), all visible bands must represent modified forms. The 150 kDa band runs together with one of the modified forms of variant CD44 which is expressed in the metastasing cells BSp73ASML. BSp73AS or simulation-transferred BSp73ASpSVneo do not possess this protein. As in BSp73ASML cells, the epitope of the cells expressed by the transfects lies freely on the cell surface.

In order to demonstrate that the expression of variant CD44 suffices in order to impart a metastatic potential to BSp73AS cells, transfects were injected into syngenic BDX rats (spontaneous metastasis protocol). In earlier experiments, metastatic tumour cells BSp73ASML spread out quickly from the place of the injection and were completely distributed about 10 days after the injection (Matzku, 1984). All local tumours were, therefore, removed by amputations on the 10th day. All carriers of BSp73ASML cells and all animals which had been injected with an overexpressing transfect developed lung metastases (Table 1). The course of the metastasis formation was comparably quick within 5-8 weeks after the injection. Animals which had received Bsp73AS cells or simulation transfects were, after this time, completely healthy (apart from due to the amputation) and even after 5 months no metastases could be ascertained.

In spite of a surprising similarity in the strong metastasis formation, there are some interesting differences. In all animals, BSp73ASML cells reach the lymph nodes and lead to a massive enlargement of various nodes in the region of the inguinal groin and next to the aorta (Table 1). A transfect (BSp73AS-pSV meta-1-14) causes lymph node enlargement in 3 of 8 animals although all animals develop lung metastases (Table 1). No lymph node enlargement is ascertainable with the other transfect (BSp73AS-pSV Meta-1-15). The transfects appear, therefore, to be able to form colonies in the lungs without an obligatory growth phase of the lymph nodes.

The experiment according to Table 1 further points to another difference between BSp73AS transfects and BSp73ASML. The individual lung metastases are macroscopically visible, whereas those of BSp73ASML are small and numerous but, in a larger series with BSp73ASML (Reber et al., 1990), 11 of 20 animals develop 5-20 larger nodes per lung than the transfects.

In order to ascertain that the metastases formed were brought about by the injected transfects and in order to exclude the improbable possibility of a spontaneous mutation, which transfers a metastatic potential, the epitope-positive proteins in the total lung extracts and in the extracts of recultured metastasis-producing cells were determined. The 150 kDa glycoprotein is detectable in the whole lung extract, as well as in the extracts of a specific lung node from an animal which has received BSpAS-pSV meta-1-15 transfects. In the case of in vitro growth, the G418-resistant strain expresses a protein of the same apparent molecular weight.

Diagnosis and Therapy

1. Analysis of human tumour material by in situ hybridisation with the human pMeta-1 sequence present. These experiments are considered as preliminary experiments before an Ab is available which recognises the human ECR.

2. Production of antibodies against the human ECR. Cloning of the human pMeta-1 sequences in bacterial expression vectors so that fusions arise with β-galactosidase or tryptophane E-product. Immunisation of rabbits with these fusion proteins or with synthesised peptides from the ECR (coupled to carrier molecules). Isolation of the polyvalent or monospecific antibodies.

Possibilities of Use:

Immunohistological investigations of clinical tumour material (diagnosis)

Detection of soluble ECR in the serum of patients with the help of ELISA tests (diagnosis)

Construction of toxin-coupled antibodies in order, with the help of the antibody, to bring the toxin into the tumour/metastasis region (therapy)

Construction of antibodies with two definite antigen binding positions. By means of this double specificity, the attempt is to be made to initiate cytotoxic reactions in the metastasis region (e.g. anti CD2 or CD3 coupling) (therapy).

3. Production of hMeta-1 protein by transfection of human or rat cells with an expression vector which carries the complete hMeta-1 cDNA sequence; or purification from LCLC97 cells.

Possibilities of Use:

Injection of the protein or parts thereof in order to block the tissue binding positions of the tumour cells

After characterisation of the binding positions, a use for therapy would also be conceivable which could depend upon the injection of large amounts of binding protein which would then block the migrating tumour cells.

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Neri, A., Welch, D., Kawaguchi, T. and Nicolson, G. L. (1982), Development and biologic properties of malignant cell sublines and clones of spontaneously metastasizing rat mammary adenocarcinoma, J. Natl. Cancer Inst. 68: 507-517.

Nicolson, G. L. (1987), Tumor cell instability, diversification, and progression to the metastatic phenotype; from oncogene to oncofetal expression, Cancer Res. 47: 1473-1487.

Nottenburg, C., Rees, G. and St. John, T. (1989), Isolation of mouse CD44 cDNA: structural features are distinct from the primate cDNA, Proc. Natl. Acad. Sci. U.S.A. 86: 8521-8525.

Stamenkovic, I., Amiot, M., Pesando, J. M. and Seed, B. (1989), A lymphocyte molecule implicated in lymph node homing is a member of the cartilage link protein family, Cell 56: 1057-1062.

Stanley K. K. and Luzio, J. P. (1984), Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins, EMBO J. 3: 1429-1434.

Wenzel, A. (1986), Charakterisierung von Differenzierungsantigenen auf dem Rattentumor Bsp 73 mit Hilfe monoklonaler Antikorper (Characterisation of differentiation antigens on the rat tumour Bsp 73 with the help of monoclonal antibodies), Diploma Dissertation, University of Karlsruhe.

Zhou, D. F. H., Ding, J. F., Picker, L. F., Bargatze, R. F., Butcher, E. C. and Goeddel, D. V. (1989), Molecular cloning and expression of Pgp-1--The mouse homolog of the human H-CAM (Hermes) lymphocyte homing receptor, J. Immunol. 143: 3390-3395.

                  TABLE 1                                                          ______________________________________                                         Metastatic spreading out of BSp73AS cells which express                        variant CD44 cDNA pMeta-1***                                                           local distribution in the case                                                 appear-                                                                              of metastatic autopsy                                            tumour clone                                                                             ance    *LN ing    *LN par                                                                               lung                                       ______________________________________                                         BSp73ASML 0/8     8/8        8/8    8/8                                                          φ 1.5-2.5**                                                                           φ 2.5-5.0                                                                         miliary                                    BSp73AS-  0/8     3/8        3/8    8/8                                        pSVMeta-1-14      φ 0.3-1.2                                                                             φ 1.0-4.5                                                                         multiple                                                                       φ 0.3-5.0                              BSp73AS-  0/8     0/8        0/8    8/8                                        pSVMeta-1-15                        5-20                                                                           φ 0.3-10.0                             BSp73AS   1/8     0/8        0/8    0/8                                        BSp73AS-  0/8     0/8        0/8    0/8                                        pSVneo                                                                         ______________________________________                                          **average diameter in mm                                                       ***the Table gives the stage 60 days after injection of the given cells        *LN = lymph nodes                                                        

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 8                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3207 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: p-Meta-1                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 113..1624                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        CTCATTGCCCAGCAGCCCCCAGCCAGTGACAGGTTCCATTCACCCTCTTTGCCCCTTCCC60                 CCGCGACCCTTTTCCAGAGGCTACTAGATCCTTTGGTTTCATCCTGCACATCATG115                     Met                                                                            GACAAGGTTTGGTGGCACACAGCTTGGGGACTACTTTGCCTCTTACAG163                            AspLysValTrpTrpHisThrAlaTrpGlyLeuLeuCysLeuLeuGln                               51015                                                                          TTGAGCCTGGCACAGCAGCAGATCGATTTGAATATAACCTGCCGTTAC211                            LeuSerLeuAlaGlnGlnGlnIleAspLeuAsnIleThrCysArgTyr                               202530                                                                         GCAGGTGTATTCCATGTGGAGAAAAATGGCCGCTACAGTATCTCCAGG259                            AlaGlyValPheHisValGluLysAsnGlyArgTyrSerIleSerArg                               354045                                                                         ACTGAAGCAGCTGACCTCTGCGAGGCTTTCAACACCACCTTGCCCACC307                            ThrGluAlaAlaAspLeuCysGluAlaPheAsnThrThrLeuProThr                               50556065                                                                       ATGGCTCAGATGGAGTTAGCCCTGAGAAAGGGGTTTGAAACATGCAGG355                            MetAlaGlnMetGluLeuAlaLeuArgLysGlyPheGluThrCysArg                               707580                                                                         TATGGGTTCATAGAAGGACACGTGGTAATCCCGAGGATCCACCCCAAC403                            TyrGlyPheIleGluGlyHisValValIleProArgIleHisProAsn                               859095                                                                         GCTATCTGTGCAGCCAACAACACAGGAGTGTATATCCTCCTCGCATCC451                            AlaIleCysAlaAlaAsnAsnThrGlyValTyrIleLeuLeuAlaSer                               100105110                                                                      AACACCTCCCACTATGACACATATTGCTTCAATGCCTCAGCTCCTCTT499                            AsnThrSerHisTyrAspThrTyrCysPheAsnAlaSerAlaProLeu                               115120125                                                                      GAAGAAGACTGTACATCAGTCACAGACCTACCCAATTCCTTCGATGGA547                            GluGluAspCysThrSerValThrAspLeuProAsnSerPheAspGly                               130135140145                                                                   CCAGTTACCATAACTATTGTCAACCGTGATGGCACCCGCTACAGCAAG595                            ProValThrIleThrIleValAsnArgAspGlyThrArgTyrSerLys                               150155160                                                                      AAGGGCGAGTATAGAACACACCAAGAAGACATCGATGCCTCAAACATT643                            LysGlyGluTyrArgThrHisGlnGluAspIleAspAlaSerAsnIle                               165170175                                                                      ATAGATGAGGATGTCAGCAGTGGATCCACCATTGAGAAGAGCACCCCA691                            IleAspGluAspValSerSerGlySerThrIleGluLysSerThrPro                               180185190                                                                      GAAGGCTACATTTTGCACACCGACCTTCCCACTTCACAGCCTACTGGA739                            GluGlyTyrIleLeuHisThrAspLeuProThrSerGlnProThrGly                               195200205                                                                      GACCGGGATGACGCCTTCTTTATTGGGAGCACCCTGGCCACCATTGCA787                            AspArgAspAspAlaPhePheIleGlySerThrLeuAlaThrIleAla                               210215220225                                                                   ACTACTCCATGGGTTTCTGCCCACACAAAACAGAACCAGGAACGGACC835                            ThrThrProTrpValSerAlaHisThrLysGlnAsnGlnGluArgThr                               230235240                                                                      CAGTGGAACCCGATCCATTCAAACCCAGAAGTACTACTTCAGACAACC883                            GlnTrpAsnProIleHisSerAsnProGluValLeuLeuGlnThrThr                               245250255                                                                      ACCAGGATGACTGATATAGACAGAAACAGCACCAGTGCTCATGGAGAA931                            ThrArgMetThrAspIleAspArgAsnSerThrSerAlaHisGlyGlu                               260265270                                                                      AACTGGACCCAGGAACCACAGCCTCCTTTCAATAACCATGAGTATCAG979                            AsnTrpThrGlnGluProGlnProProPheAsnAsnHisGluTyrGln                               275280285                                                                      GATGAAGAGGAGACCCCACATGCTACAAGCACAACCTGGGCAGATCCT1027                           AspGluGluGluThrProHisAlaThrSerThrThrTrpAlaAspPro                               290295300305                                                                   AATAGCACAACAGAAGAAGCAGCTACCCAGAAGGAGAAGTGGTTTGAG1075                           AsnSerThrThrGluGluAlaAlaThrGlnLysGluLysTrpPheGlu                               310315320                                                                      AATGAATGGCAGGGGAAGAACCCACCCACCCCAAGTGAAGACTCCCAT1123                           AsnGluTrpGlnGlyLysAsnProProThrProSerGluAspSerHis                               325330335                                                                      GTGACAGAAGGGACAACTGCCTCAGCCCACAACAACCATCCAAGTCAA1171                           ValThrGluGlyThrThrAlaSerAlaHisAsnAsnHisProSerGln                               340345350                                                                      AGAATGACAACACAGAGTCAAGAGGATGTTTCATGGACCGATTTCTTC1219                           ArgMetThrThrGlnSerGlnGluAspValSerTrpThrAspPhePhe                               355360365                                                                      GACCCAATCTCACATCCAATGGGACAAGGTCATCAAACAGAAAGCAAG1267                           AspProIleSerHisProMetGlyGlnGlyHisGlnThrGluSerLys                               370375380385                                                                   GGACACTCAAGTGGGAATCAAGACAGTGGAGTGACCACAACTTCTGGT1315                           GlyHisSerSerGlyAsnGlnAspSerGlyValThrThrThrSerGly                               390395400                                                                      CCTGCGAGGAGACCTCAGATTCCAGAGTGGCTTATCATCTTGGCATCC1363                           ProAlaArgArgProGlnIleProGluTrpLeuIleIleLeuAlaSer                               405410415                                                                      CTCCTGGCGCTGGCTCTGATTCTTGCCGTCTGCATTGCTGTCAACAGT1411                           LeuLeuAlaLeuAlaLeuIleLeuAlaValCysIleAlaValAsnSer                               420425430                                                                      AGGAGAAGGTGTGGGCAGAAGAAGAAGCTGGTGATCAACAGTGGCAAT1459                           ArgArgArgCysGlyGlnLysLysLysLeuValIleAsnSerGlyAsn                               435440445                                                                      GGAACAGTGGAAGACAGGAAACCAAGTGAACTCAACGGGGAGGCCAGC1507                           GlyThrValGluAspArgLysProSerGluLeuAsnGlyGluAlaSer                               450455460465                                                                   AAGTCTCAGGAAATGGTGCATTTGGTGAACAAGGAACCAACAGAGACT1555                           LysSerGlnGluMetValHisLeuValAsnLysGluProThrGluThr                               470475480                                                                      CCGGACCAGTTTATGACAGCTGATGAGACCCGGAATCTGCAGAGTGTG1603                           ProAspGlnPheMetThrAlaAspGluThrArgAsnLeuGlnSerVal                               485490495                                                                      GATATGAAGATTGGGGTGTAGTGCCTATGCCACTAACTTGAAAAGACA1651                           AspMetLysIleGlyVal                                                             500                                                                            CAACAATTGGAGACATGTCATTACTGGGAGCTGGGACCCTTAACAGATGCAATGTGCTAC1711               TGATTATTTTTTATTGGGATTATTTTGGGCATAAAATTTCCCTTTTTTTGTTTTTTAAAA1771               GTTTGTTTTCCAATTTATGAAAATAGCATTGCTTTCTGAAATGAGGGTCTCTTCCAGTTC1831               CTCCTTAGAGGCCTTGCATTACCAGGGTATGCTACCATAGGCTTCTACCAAATGAATACT1891               CTTGGTCCCGATTGAACCCAAAGTCCCAGGTAACATCCACCAGCTAAGGATTTCCCCAGA1951               ACTTAGAGAGATTGGTCTCTGGGAGGAAATTTGAATGGGTCCATATTGCCTCCCAGCAGT2011               CCAATCTGTAGGCATTGCTTTGCAGTGGATGGGAGATCAGGTGTACTGGTTACACACTCT2071               CTTTATAGACTCCCTTCTGCTGGAAAATTTCCACATGCTTCTGAGAGATTCCCCAAAGGT2131               GACGCTATTTATCTTTAGTAAGCTATTTATCTTTGTTTTTGAAATATCAAACCCTGGAGG2191               TCCTTTTTTCAGTATGACTTTTTTTATTTTGTTTTTTTTTATTTTGTTTTTTAGGTTACT2251               TTGTCAGAAGCATAACAGGGTATAAGTTGATTCATAATAAATACCTGTCCATCTTCCATC2311               TTGACCTGTTGTGCTGTGATCCTTCAGTTTCTAAATCAGCAAGGTCTGAGTCTTTGTAGC2371               ACATCAATGTGACCTTAGTATGGTCCTCTGAAACTCATGTTAGAGCATCCGTGCCCTGCT2431               TGGGTTTACCCAGCTGAATCTCAGAAGATCAAGGACAGGAGCACTGTTTTCATTCTAGGA2491               CTATCAAAGGGGTTTCTCTCCTGTTCAAGAATCTGAATTGGGAGTAGGAGAGCTTCTGTC2551               CCTTTTATGTTTCGATAACCACCCATTTCTCTTTCTTAAAGGGCACATTAAGTTTTTATA2611               TCTTACAACATTCGCGGTCCTGTTTCATAGACACTGATCTTATTGGCACTTTCACAAAAC2671               AGTGTGGAGGGGACTTCTGACACCTTATAGTAAAAGGAGAAGCCAACAGAAATGAAAGTG2731               TGGACAGAGAGCAGTAGATTGGCATGAGGAGGCATGATGTACAACCCCCAGACCACTCTT2791               TCCATCACCACATTTGTTGATGCTTTCGCAAGCCAGTTGGTACTTAGAATCAGTTCCCCA2851               GGGAATCCTTCAAAAAGCCATAAGAATGCCCACCCCTGGAATCTTACCACCACCAGATGA2911               GCAGGTTTATGGTTTAGCAAAAGGAGAATGCTGTCACCCTCTGACCTCATAGTTTTCACA2971               TACTGGGCAAGTGTTCATCTGCCAGGATGCCCCATTGCTCCTAGGTCTTCCCAGGTACCT3031               TGTAGAAGAACTTAAATCTATAAAATAAGGCTTTCTCTAAAATGGAACTTCCTTTCTAAG3091               GCTCCCATTTTTACTGTTGACTAAATTTATATGTTTAATAGTTTTTTTTCAAATAAAAAC3151               AAACACAAAAAGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA3207                   (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 503 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        MetAspLysValTrpTrpHisThrAlaTrpGlyLeuLeuCysLeuLeu                               151015                                                                         GlnLeuSerLeuAlaGlnGlnGlnIleAspLeuAsnIleThrCysArg                               202530                                                                         TyrAlaGlyValPheHisValGluLysAsnGlyArgTyrSerIleSer                               354045                                                                         ArgThrGluAlaAlaAspLeuCysGluAlaPheAsnThrThrLeuPro                               505560                                                                         ThrMetAlaGlnMetGluLeuAlaLeuArgLysGlyPheGluThrCys                               65707580                                                                       ArgTyrGlyPheIleGluGlyHisValValIleProArgIleHisPro                               859095                                                                         AsnAlaIleCysAlaAlaAsnAsnThrGlyValTyrIleLeuLeuAla                               100105110                                                                      SerAsnThrSerHisTyrAspThrTyrCysPheAsnAlaSerAlaPro                               115120125                                                                      LeuGluGluAspCysThrSerValThrAspLeuProAsnSerPheAsp                               130135140                                                                      GlyProValThrIleThrIleValAsnArgAspGlyThrArgTyrSer                               145150155160                                                                   LysLysGlyGluTyrArgThrHisGlnGluAspIleAspAlaSerAsn                               165170175                                                                      IleIleAspGluAspValSerSerGlySerThrIleGluLysSerThr                               180185190                                                                      ProGluGlyTyrIleLeuHisThrAspLeuProThrSerGlnProThr                               195200205                                                                      GlyAspArgAspAspAlaPhePheIleGlySerThrLeuAlaThrIle                               210215220                                                                      AlaThrThrProTrpValSerAlaHisThrLysGlnAsnGlnGluArg                               225230235240                                                                   ThrGlnTrpAsnProIleHisSerAsnProGluValLeuLeuGlnThr                               245250255                                                                      ThrThrArgMetThrAspIleAspArgAsnSerThrSerAlaHisGly                               260265270                                                                      GluAsnTrpThrGlnGluProGlnProProPheAsnAsnHisGluTyr                               275280285                                                                      GlnAspGluGluGluThrProHisAlaThrSerThrThrTrpAlaAsp                               290295300                                                                      ProAsnSerThrThrGluGluAlaAlaThrGlnLysGluLysTrpPhe                               305310315320                                                                   GluAsnGluTrpGlnGlyLysAsnProProThrProSerGluAspSer                               325330335                                                                      HisValThrGluGlyThrThrAlaSerAlaHisAsnAsnHisProSer                               340345350                                                                      GlnArgMetThrThrGlnSerGlnGluAspValSerTrpThrAspPhe                               355360365                                                                      PheAspProIleSerHisProMetGlyGlnGlyHisGlnThrGluSer                               370375380                                                                      LysGlyHisSerSerGlyAsnGlnAspSerGlyValThrThrThrSer                               385390395400                                                                   GlyProAlaArgArgProGlnIleProGluTrpLeuIleIleLeuAla                               405410415                                                                      SerLeuLeuAlaLeuAlaLeuIleLeuAlaValCysIleAlaValAsn                               420425430                                                                      SerArgArgArgCysGlyGlnLysLysLysLeuValIleAsnSerGly                               435440445                                                                      AsnGlyThrValGluAspArgLysProSerGluLeuAsnGlyGluAla                               450455460                                                                      SerLysSerGlnGluMetValHisLeuValAsnLysGluProThrGlu                               465470475480                                                                   ThrProAspGlnPheMetThrAlaAspGluThrArgAsnLeuGlnSer                               485490495                                                                      ValAspMetLysIleGlyVal                                                          500                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1062 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: human cDNA                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..1062                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        GTACACCCCATCCCAGACGAAGACAGTCCCTGGATCACCGACAGCACA48                             ValHisProIleProAspGluAspSerProTrpIleThrAspSerThr                               151015                                                                         GACAGAATCCCTGCTACCACAGGCTGGGAGCCAAATGAAGAAAATGAA96                             AspArgIleProAlaThrThrGlyTrpGluProAsnGluGluAsnGlu                               202530                                                                         GATGAAAGAGACAGACACCTCAGTTTTTCTGGATCAGGCATTGATGAT144                            AspGluArgAspArgHisLeuSerPheSerGlySerGlyIleAspAsp                               354045                                                                         GATGAAGATTTTATCTCCAGCACCATTTCAACCACACCACGGGCCTTT192                            AspGluAspPheIleSerSerThrIleSerThrThrProArgAlaPhe                               505560                                                                         GACCACACAAAACAGAACCAGGACTGGACCCAGTGGAACCCAAGCCAT240                            AspHisThrLysGlnAsnGlnAspTrpThrGlnTrpAsnProSerHis                               65707580                                                                       TCAAATCCGGAAGTGCTACTTCAGACAACCACAAGGATGACTGATGTA288                            SerAsnProGluValLeuLeuGlnThrThrThrArgMetThrAspVal                               859095                                                                         GACAGAAATGGCACCACTGCTTATGAAGGAAACTGGAACCCAGAAGCA336                            AspArgAsnGlyThrThrAlaTyrGluGlyAsnTrpAsnProGluAla                               100105110                                                                      CACCCTCCCCTCATTCACCATGAGCATCATGAGGAAGAAGAGACCCCA384                            HisProProLeuIleHisHisGluHisHisGluGluGluGluThrPro                               115120125                                                                      CATTCTACAAGCACAATCCAGGCAACTCCTAGTAGTACAACGGAAGAA432                            HisSerThrSerThrIleGlnAlaThrProSerSerThrThrGluGlu                               130135140                                                                      ACAGCTACCCAGAAGGAACAGTGGTTTGGCAACAGATGGCATGAGGGA480                            ThrAlaThrGlnLysGluGlnTrpPheGlyAsnArgTrpHisGluGly                               145150155160                                                                   TATCGCCAAACACCCAGAGAAGACTCCCATTCGACAACAGGGACAGCT528                            TyrArgGlnThrProArgGluAspSerHisSerThrThrGlyThrAla                               165170175                                                                      GCAGCCTCAGCTCATACCAGCCATCCAATGCAAGGAAGGACAACACCA576                            AlaAlaSerAlaHisThrSerHisProMetGlnGlyArgThrThrPro                               180185190                                                                      AGCCCAGAGGACAGTTCCTGGACTGATTTCTTCAACCCAATCTCACAC624                            SerProGluAspSerSerTrpThrAspPhePheAsnProIleSerHis                               195200205                                                                      CCCATGGGACGAGGTCATCAAGCAGGAAGAAGGATGGATATGGACTCC672                            ProMetGlyArgGlyHisGlnAlaGlyArgArgMetAspMetAspSer                               210215220                                                                      AGTCATAGTACAACGCTTCAGCCTACTGCAAATCCAAACACAGGTTTG720                            SerHisSerThrThrLeuGlnProThrAlaAsnProAsnThrGlyLeu                               225230235240                                                                   GTGGAAGATTTGGACAGGACAGGACCTCTTTCAATGACAACGCAGCAG768                            ValGluAspLeuAspArgThrGlyProLeuSerMetThrThrGlnGln                               245250255                                                                      AGTAATTCTCAGAGCTTCTCTACATCACATGAAGGCTTGGAAGAAGAT816                            SerAsnSerGlnSerPheSerThrSerHisGluGlyLeuGluGluAsp                               260265270                                                                      AAAGACCATCCAACAACTTCTACTCTGACATCAAGCAATAGGAATGAT864                            LysAspHisProThrThrSerThrLeuThrSerSerAsnArgAsnAsp                               275280285                                                                      GTCACAGGTGGAAGAAGAGACCCAAATCATTCTGAAGGCTCAACTACT912                            ValThrGlyGlyArgArgAspProAsnHisSerGluGlySerThrThr                               290295300                                                                      TTACTGGAAGGTTATACCTCTCATTACCCACACACGAAGGAAAGCAGG960                            LeuLeuGluGlyTyrThrSerHisTyrProHisThrLysGluSerArg                               305310315320                                                                   ACCTTCATCCCAGTGACCTCAGCTAAGACTGGGTCCTTTGGAGTTACT1008                           ThrPheIleProValThrSerAlaLysThrGlySerPheGlyValThr                               325330335                                                                      GCAGTTACTGTTGGAGATTCCAACTCTAATGTCAATCGTTCCTTATCA1056                           AlaValThrValGlyAspSerAsnSerAsnValAsnArgSerLeuSer                               340345350                                                                      GGAGAC1062                                                                     GlyAsp                                                                         (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 354 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        ValHisProIleProAspGluAspSerProTrpIleThrAspSerThr                               151015                                                                         AspArgIleProAlaThrThrGlyTrpGluProAsnGluGluAsnGlu                               202530                                                                         AspGluArgAspArgHisLeuSerPheSerGlySerGlyIleAspAsp                               354045                                                                         AspGluAspPheIleSerSerThrIleSerThrThrProArgAlaPhe                               505560                                                                         AspHisThrLysGlnAsnGlnAspTrpThrGlnTrpAsnProSerHis                               65707580                                                                       SerAsnProGluValLeuLeuGlnThrThrThrArgMetThrAspVal                               859095                                                                         AspArgAsnGlyThrThrAlaTyrGluGlyAsnTrpAsnProGluAla                               100105110                                                                      HisProProLeuIleHisHisGluHisHisGluGluGluGluThrPro                               115120125                                                                      HisSerThrSerThrIleGlnAlaThrProSerSerThrThrGluGlu                               130135140                                                                      ThrAlaThrGlnLysGluGlnTrpPheGlyAsnArgTrpHisGluGly                               145150155160                                                                   TyrArgGlnThrProArgGluAspSerHisSerThrThrGlyThrAla                               165170175                                                                      AlaAlaSerAlaHisThrSerHisProMetGlnGlyArgThrThrPro                               180185190                                                                      SerProGluAspSerSerTrpThrAspPhePheAsnProIleSerHis                               195200205                                                                      ProMetGlyArgGlyHisGlnAlaGlyArgArgMetAspMetAspSer                               210215220                                                                      SerHisSerThrThrLeuGlnProThrAlaAsnProAsnThrGlyLeu                               225230235240                                                                   ValGluAspLeuAspArgThrGlyProLeuSerMetThrThrGlnGln                               245250255                                                                      SerAsnSerGlnSerPheSerThrSerHisGluGlyLeuGluGluAsp                               260265270                                                                      LysAspHisProThrThrSerThrLeuThrSerSerAsnArgAsnAsp                               275280285                                                                      ValThrGlyGlyArgArgAspProAsnHisSerGluGlySerThrThr                               290295300                                                                      LeuLeuGluGlyTyrThrSerHisTyrProHisThrLysGluSerArg                               305310315320                                                                   ThrPheIleProValThrSerAlaLysThrGlySerPheGlyValThr                               325330335                                                                      AlaValThrValGlyAspSerAsnSerAsnValAsnArgSerLeuSer                               340345350                                                                      GlyAsp                                                                         (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 355 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: rat protein                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        SerGlnProThrGlyAspArgAspAspAlaPhePheIleGlySerThr                               151015                                                                         LeuAlaThrSerThrGluSerAsnThrAsnProThrGlyTrpLysPro                               202530                                                                         AsnGluGluAsnGluAspGluThrAspLysTyrProAsnPheSerGly                               354045                                                                         SerGlyIleAspAspAspGluAspPheIleSerSerThrIleAlaThr                               505560                                                                         ThrProTrpValSerAlaHisThrLysGlnAsnGlnGluArgThrGln                               65707580                                                                       TrpAsnProIleHisSerAsnProGluValLeuLeuGlnThrThrThr                               859095                                                                         ArgMetThrAspIleAspArgAsnSerThrSerAlaHisGlyGluAsn                               100105110                                                                      TrpThrGlnGluProGlnProProPheAsnAsnHisGluTyrGlnAsp                               115120125                                                                      GluGluGluThrProHisAlaThrSerThrThrTrpAlaAspProAsn                               130135140                                                                      SerThrThrGluGluAlaAlaThrGlnLysGluLysTrpPheGluAsn                               145150155160                                                                   GluTrpGlnGlyLysAsnProProThrProSerGluAspSerHisVal                               165170175                                                                      ThrGluGlyThrThrAlaSerAlaHisAsnAsnHisProSerGlnArg                               180185190                                                                      MetThrThrGlnSerGlnGluAspValSerTrpThrAspPhePheAsp                               195200205                                                                      ProIleSerHisProMetGlyGlnGlyHisGlnThrGluSerLysAsp                               210215220                                                                      ThrGlySerSerHisSerThrThrLeuGlnProThrAlaAlaProAsn                               225230235240                                                                   ThrHisLeuValGluAspLeuAsnArgThrGlyProLeuSerValThr                               245250255                                                                      ThrProGlnSerHisSerGlnAsnPheSerThrLeuProGlyGluLeu                               260265270                                                                      GluGluGlyGluAspHisProThrThrSerValLeuProSerSerThr                               275280285                                                                      LysSerGlyArgArgArgGlyGlySerLeuProArgAspThrThrThr                               290295300                                                                      SerLeuGluGlyTyrThrProGlnTyrProAspThrMetGluAsnGly                               305310315320                                                                   ThrLeuPheProValThrProAlaLysThrGluValPheGlyGluThr                               325330335                                                                      GluGlyThrValAlaThrAspSerAsnPheAsnValAspGlySerLeu                               340345350                                                                      ProGlyAsp                                                                      355                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 361 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: hCD44                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        MetAspLysPheTrpTrpHisAlaAlaTrpGlyLeuCysLeuValPro                               151015                                                                         LeuSerLeuAlaGlnIleAspLeuAsnIleThrCysArgPheAlaGly                               202530                                                                         ValPheHisValGluLysAsnGlyArgTyrSerIleSerArgThrGlu                               354045                                                                         AlaAlaAspLeuCysLysAlaPheAsnSerThrLeuProThrMetAla                               505560                                                                         GlnMetGluLysAlaLeuSerIleGlyPheGluThrCysArgTyrGly                               65707580                                                                       PheIleGluGlyHisValValIleProArgIleHisProAsnSerIle                               859095                                                                         CysAlaAlaAsnAsnThrGlyValTyrIleLeuThrTyrAsnThrSer                               100105110                                                                      GlnTyrAspThrTyrCysPheAsnAlaSerAlaProProGluGluAsp                               115120125                                                                      CysThrSerValThrAspLeuProAsnAlaPheAspGlyProIleThr                               130135140                                                                      IleThrIleValAsnArgAspGlyThrArgTyrValGlnLysGlyGlu                               145150155160                                                                   TyrArgThrAsnProGluAspIleTyrProSerAsnProThrAspAsp                               165170175                                                                      AspValSerSerGlySerSerSerGluArgSerSerThrSerGlyGly                               180185190                                                                      TyrIlePheTyrThrPheSerThrValHisProIleProAspGluAsp                               195200205                                                                      SerProTrpIleThrAspSerThrAspArgIleProAlaThrArgAsp                               210215220                                                                      GlnAspThrPheHisProSerGlyGlySerHisThrThrHisGluSer                               225230235240                                                                   GluSerAspGlyHisSerHisGlySerGlnGluGlyGlyAlaAsnThr                               245250255                                                                      ThrSerGlyProIleArgThrProGlnIleProGluTrpLeuIleIle                               260265270                                                                      LeuAlaSerLeuLeuAlaLeuAlaLeuIleLeuAlaValCysIleAla                               275280285                                                                      ValAsnSerArgArgArgCysGlyGlnLysLysLysLeuValIleAsn                               290295300                                                                      SerGlyAsnGlyAlaValGluAspArgLysProSerGlyLeuAsnGly                               305310315320                                                                   GluAlaSerLysSerGlnGluMetValHisLeuValAsnLysGluSer                               325330335                                                                      SerGluThrProAspGlnPheMetThrAlaAspGluThrArgAsnLeu                               340345350                                                                      GlnAsnValAspMetLysIleGlyVal                                                    355360                                                                         (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 363 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: mCD44                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        MetAspLysPheTrpTrpHisThrAlaTrpGlyLeuCysLeuLeuGln                               151015                                                                         LeuSerLeuAlaHisGlnGlnIleAspLeuAsnValThrCysArgTyr                               202530                                                                         AlaGlyValPheCysValGluLysAsnGlyArgTyrSerIleSerArg                               354045                                                                         ThrGluAlaAlaAspLeuCysGlnAlaPheAsnSerThrLeuProThr                               505560                                                                         MetAspGlnMetLysLeuAlaLeuSerLysGlyPheGluThrCysArg                               65707580                                                                       TyrGlyPheIleGluGlyAsnValValIleProArgIleHisProAsn                               859095                                                                         AlaIleCysAlaAlaAsnHisThrGlyValTyrIleLeuValThrSer                               100105110                                                                      AsnThrSerHisTyrAspThrTyrCysPheAsnAlaSerAlaProPro                               115120125                                                                      GluGluAspCysThrSerValThrAspLeuProAsnSerPheAspGly                               130135140                                                                      ProValThrIleThrIleValAsnArgAspGlyThrArgTyrSerLys                               145150155160                                                                   LysGlyGluTyrArgThrHisGlnGluAspIleAspAlaSerAsnIle                               165170175                                                                      IleAspAspAspValSerSerGlySerThrIleGluLysSerThrPro                               180185190                                                                      GluGlyTyrIleLeuHisThrTyrLeuProThrGluGlnProThrGly                               195200205                                                                      AspGlnAspAspSerPhePheIleArgSerThrLeuAlaThrArgAsp                               210215220                                                                      ArgAspSerSerLysAspSerArgGlySerSerArgThrValThrHis                               225230235240                                                                   GlySerGluLeuAlaGlyHisSerSerAlaAsnGlnAspSerGlyVal                               245250255                                                                      ThrThrThrSerGlyProMetArgArgProGlnIleProGluTrpLeu                               260265270                                                                      IleIleLeuAlaSerLeuLeuAlaLeuAlaLeuIleLeuAlaValCys                               275280285                                                                      IleAlaValAsnSerArgArgArgCysGlyGlnLysLysLysLeuVal                               290295300                                                                      IleAsnGlyGlyAsnGlyThrValGluAspArgLysProSerGluLeu                               305310315320                                                                   AsnGlyGluAlaSerLysSerGlnGluMetValHisLeuValAsnLys                               325330335                                                                      GluProSerGluThrProAspGlnCysMetThrAlaAspGluThrArg                               340345350                                                                      AsnLeuGlnSerValAspMetLysIleGlyVal                                              355360                                                                         (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: rCD44                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        SerAspGlyAspSerSerMetAspProArgGlyGlyPheAspThrVal                               151015                                                                         ThrHisGlySerGluLeuAla                                                          20                                                                             __________________________________________________________________________ 

We claim:
 1. An isolated Meta-1 surface polypeptide of metastasizing tumor cells comprisingan amino acid sequence selected from the group consisting of:amino acid residues 224 to 378 of SEQ ID NO:2, amino acid residues 53 to 219 of SEQ ID NO:4 and amino acid residues 77 to 186 of SEQ ID NO:4.
 2. An isolated Meta-1 surface polypeptide comprising an amino acid sequence having SEQ ID No:
 2. 3. An isolated Meta-1 surface polypeptide comprising an amino acid sequence having SEQ ID No:
 4. 